Follicle-stimulating hormone (FSH) is a glycoprotein having a molecular weight of 34 kD, which consists of two subunits, α and β, and has an activity to promote production of estrogen in the ovary and its secretion from it. Medical drugs containing human FSH as their principal agent are used in infertility treatment. At first, the human FSH contained in them a protein which was obtained through purification from human urine. Recently, recombinant human FSH produced making use of the genetic recombination technology was approved for sale and has been used as a therapeutic drug for infertility treatment with indications for “controlled ovarian stimulation for growth of multiple follicles” and “induction of ovulation in cases of anovulation or infrequent ovulation accompanying hypothalamic-pituitary dysfunction”.
The genes encoding the α- and the β-subunits of human FSH have been cloned, of which the α-subunit is a protein that is identical to the α-subunit of human chorionic gonadotropin (hCG) (see patent document 1 and non-patent document 1).
A method for production of recombinant human FSH has been disclosed in which mammalian cells (such as CHO cells) were employed that had been transformed with expression vectors having incorporated genes encoding one or the other of the two subunits of human FSH (see patent document 2). According to the document, the genes encoding the two subunits of human FSH were separately incorporated into different expression vectors, which then were introduced into the same CHO cells. And, the two subunits, separately expressed by those expression vectors, form a hetero dimer within the cell, thereby giving active human FSH. However, the document contains no specific description as to how the human FSH is purified.
A method for purification of recombinant human FSH has been disclosed in a publication, in which culture supernatant of recombinant human FSH producing cells cultured in either a serum-containing or a serum-free medium, was subjected to sequential chromatography using a blue-dye, a hydrophobic, and then a reverse-phase column in the order (see patent document 3). The document also discloses a method for purification of recombinant human FSH, in which anion-exchange column chromatography was additionally employed following the above chromatography procedure. In both of these methods, 2-propanol is used to elute FSH from the reverse-phase column. Organic solvents such as 2-propanol, however, may denature proteins, and, further, their use in an industrial-scale production is economically disadvantageous, for such organic solvents are unfavorable to the environment and therefore require a facility in which to treat the waste fluid containing them.
Further, a method for purification of recombinant human FSH has been disclosed in a publication, in which chromatography using an anion-exchange, an immobilized metal ion adsorption, a hydrophobic, and then a reverse phase column is employed in the order (see patent document 4). As it employs 2-propanol, too, to elute the recombinant human FSH from the reverse-phase column, this method is not desirable either to the environment or in the economic sense for the same reason as is mentioned above. Furthermore, though it is for urine-derived human FSH, another method has also been disclosed in a publication in which purification of human FSH is performed by chromatography using an anti-human FSH antibody affinity column and then a reverse-phase column (see patent document 5). This method, however, also has the same disadvantage as in those methods mentioned above, for it also employs 2-propanol to elute human FSH from a reverse-phase column.
On the other hand, a method has been disclosed for purification of recombinant human FSH without using any organic solvent. The method utilizes chromatography employing a dye affinity, a weak ion-exchange, a hydrophobic, a strong ion-exchange, and then a hydrophobic column in the order (patent document 6). The examples presented in the document, however, are not of wild-type human FSH but only of those of mutant-type proteins which were formed by incorporating a fragment of another amino acid sequence into human wild-type FSH.
In addition, through a method has been known for purification of human FSH from human urine (patent document 7), the method involves a process in which an organic solvent (ethanol) is employed, and therefore has the same disadvantage as is mentioned above.    [Patent document 1] Japanese Patent No. 2008344    [Patent document 2] Japanese Patent No. 2559196    [Patent document 3] WO 2006/051070    [Patent document 4] WO 2005/063811    [Patent document 5] Japanese Patent No. 2523843    [Patent document 6] WO 2007/065918    [Patent document 7] Japanese Patent Application Publication No. 2001-323000    [Non-patent document 1] Nature, 286: 684-687 (1980)